GSM7977222: LB1 pA-DamID, i9, recombined, clone 14, replicate 2, dam only; Mus musculus; OTHER
One million cells were collected by centrifugation (500 g, 3 min) and washed sequentially in PBS and digitonin wash buffer (DigWash) (20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.02% digitonin, cOmplete Protease Inhibitor Cocktail). Cells were rotated for 2 h at 4 °C in 200 μL DigWash with 1:100 LaminB1 antibody (Abcam, ab16048, rabbit) or 1:100 H3K9me3 antibody (Diagenone, C15410193, Rabbit), followed by a wash step with DigWash. This was repeated with a 1:200 pA-Dam solution (~60 NEB Dam units), followed by 2 wash steps. Dam activity was induced by an incubation for 30 min at 37 °C in 100 μL DigWash supplemented with 80 μM SAM. For every condition, another 1 million cells were processed in only DigWash and during Dam activation incubated with 4 units of Dam enzyme (NEB, M0222L). This Dam only control sample serves to account for DNA accessibility and amplification biases. Genomic DNA was isolated (Bioline, bio-52067) and ~ 500 ng were digested with DpnI (10 U, NEB, R0176L) in CutSmart Buffer 1X (8h 37°C, 20 min 80°C) in a total volume of 10 µL. A-tailing was performed by addition of 5 µL of the A-tailing mix (0.5 µL of Cutsmart buffer 10X, 0.25 µL Klenow 50 U/µL (NEB, M0212M), 0.05 µL dATP 100 mM, 4.2 µL H2O) and incubation 30 min at 37°C followed by 20 min at 75°C. Adapters were ligated by adding 15 µL of the ligation mix (3 µL T4 Ligase Buffer 10X, 0.5 µL T4 ligase (5 U/µL , Roche, 10799009001), 0.25 µl of x-Gene Stubby Adapter 50 mM (IDT), 11.25 µl H2O) and incubating samples 16h at 16°C and 10 min at 65°C. Finally, the Methyl Indexed PCR was performed by mixing 4 µL of ligated DNA with x-Gen Dual combinatorial Indexes (IDT) (125 nM final concentration) and MyTaq RedMix (Bioline, BIO-25048) in a final volume of 40 µL. The following PCR program was used: 1 X (1 min 94°C), 14 X (30 sec 94°C, 30 sec 58°C, 30 sec 72°C), 1X (2 min 72°C).